Recently, novel pluripotent stem cells that are different from embryonic stem (ES) cells but are ES-cell like, i.e., induced pluripotent stem (iPS) cells, were generated from adult somatic cells by transduction with genes encoding defined transcription factors (Takahashi, K. and Yamanaka, S., Cell 126, 663-676 (2006); Okita, K., Ichisaka, T. and Yamanaka, S., Nature 448, 313-317 (2007); Takahashi, K. et al., Cell 131, 861-872 (2007); Yu, J. et al., Science 318, 1917-1920 (2007)). Establishment of iPS cells from adult human tissue is facilitating clinical application of iPS cells through cell transplantation-based regenerative medicine and generation of patient-specific cellular models and drug discovery systems (Yamanaka, S., Cell Stem Cell 1, 39-49 (2007); Laflamme, M. A. and Murry, C. E., Nat. Biotechnol. 23, 845-856 (2005); Kovacic, J. C., et al., Cell Stem Cell 1, 628-633 (2007)).
As ES cells and iPS cells show almost similar overall features for growth, pluripotency, and differentiation (Takahashi, K. and Yamanaka, S., Cell 126, 663-676 (2006); Okita, K. et al., Nature 448, 313-317 (2007); Takahashi, K. et al., Cell 131, 861-872 (2007); Yu, J. et al., Science 318, 1917-1920 (2007); Narazaki, G, et al., Circulation 118, 498-506 (2008)), studies for ES cells may be utilized as scientific and technological basis for iPS cell research. Previously, we established a novel ES cell differentiation system that can reproduce the early process of cardiovascular development in vitro (Yamashita, J. et al., Nature 408, 92-96 (2000); Yamashita, J. K. et al., FASEB J. 19, 1534-1536 (2005); Yanagi, K. et al., Stem Cells 25, 2712-2719 (2007); Yurugi-Kobayashi, T. et al., Arterioscler. Thromb. Vasc. Biol. 26, 1977-1984 (2006); Kono. T. et al., Arterioscler. Thromb. Vasc. Biol. 26, 2070-2076 (2006); Hiraoka-Kanie, M. et al, Biochem. Biophys. Res. Commun. 351, 669-674 (2006)). Endothelial cells (ECs), mural cells (MCs; pericytes and vascular smooth muscle cells) (Yamashita, J. et al., Nature 408, 92-96 (2000)), and cardiomyocytes (Yamashita, J. K. et al., FASEB J. 19, 1534-1536 (2005)) are systematically induced from common mesodermal precursor, i.e., Flk1 (also designated as vascular endothelial growth factor receptor-2 (VEGFR2))-expressing cells. We also succeeded in identifying a cardiac progenitor population, FCV cells (Flk1+/CXCR4+/vascular endothelial cadherin− cell population) among the progeny of Flk1+ mesoderm cells (Yamashita, J. K. et al., FASEB J. 19, 1534-1536 (2005)). Though FCV cells, which are only a small subset of Flk1+ cell progeny, showed highly cardiac specific progenitor activity, efficient expanding method of FCV cells remain unknown. More recently, we succeeded in establishing a similar mouse iPS differentiation system for cardiovascular cells (Narazaki, G, et al., Circulation 118, 498-506 (2008)), enabling to examine the induction of FCV progenitor cells from iPS cells.
However, as for cardiomyocytes, iPS cell technologies, such as efficient ways to induce cardiomyocytes, identification and expansion of cardiac progenitors, and establishment of model systems for cardiac differentiation, have not been established.
An object of this invention is to provide a method for effectively induce cardiac progenitor cells and/or cardiomyocytes from ES cells or iPS cells.
Another object of this invention is to provide cardiac progenitor cells or cardiomyocytes produced by said method.
Another object of this invention is to provide a method for treating a subject with heart disease by using said cardiac progenitor cells or cardiomyocytes.
Another object of this invention is to provide a use of said cardiac progenitor cells or cardiomyocytes in manufacture of a medicament or transplant or implant for treatment of a heart disease.
Another object of this invention is to provide a method for screening for an agent capable of inducing cardiomyocytes and/or cardiac progenitor cells.
We have now found that addition of cyclosporin-A (CSA), an immunosuppressant, to Flk1+ mesoderm cells potently induces cardiomyocytes through specific expansion of FCV cardiac progenitor population. We have further now found this simple method in the ES cell systems could be completely adapted for use in iPS cells, specifically that cardiac progenitors and cardiomyocytes could be effectively induced from Flk1+ mesoderm cells derived from human or mouse iPS cells. This novel differentiation technology would broadly contribute to cardiac regeneration by providing cell sources, transplantation strategies, and drug discovery.